From dynamic combinatorial ‘hit’ to lead: in vitro and in vivo activity of compounds targeting the pathogenic RNAs that cause myotonic dystrophy

Myotonic dystrophy type 1 (DM1) is a dominantly inherited neuromuscular disorder resulting from expression of RNA containing an expanded CUG repeat (CUGexp). The pathogenic RNA is retained in nuclear foci. Poly-(CUG) binding proteins in the Muscleblind-like (MBNL) family are sequestered in foci, causing misregulated alternative splicing of specific pre-mRNAs. Inhibitors of MBNL1-CUGexp binding have been shown to restore splicing regulation and correct phenotypes in DM1 models. We therefore conducted a high-throughput screen to identify novel inhibitors of MBNL1-(CUG)12 binding. The most active compound was lomofungin, a natural antimicrobial agent. We found that lomofungin undergoes spontaneous dimerization in DMSO, producing dilomofungin, whose inhibition of MBNL1–(CUG)12 binding was 17-fold more potent than lomofungin itself. However, while dilomofungin displayed the desired binding characteristics in vitro, when applied to cells it produced a large increase of CUGexp RNA in nuclear foci, owing to reduced turnover of the CUGexp transcript. By comparison, the monomer did not induce CUGexp accumulation in cells and was more effective at rescuing a CUGexp-induced splicing defect. These results support the feasibility of high-throughput screens to identify compounds targeting toxic RNA, but also demonstrate that ligands for repetitive sequences may have unexpected effects on RNA decay.

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“…Assays for luciferase activity were performed as previously described (26), 3 days after addition of vehicle or drug.…”

Section: Methodsmentioning

confidence: 99%

Lomofungin and dilomofungin: inhibitors of MBNL1-CUG RNA binding with distinct cellular effects

Hoskins

Ofori

Chen

et al. 2014

Nucleic Acids Research

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“…A variety of small molecules have been described that inhibit the (CUG) n -MBNL1 complex and improve DM1-associated molecular defects in vitro and in some cases also in vivo. Several approaches were successful in identifying small molecules, such as screening of known nucleic acid binders (21), rational design of small molecules based on the structure of (CUG) n RNA (22), rational design of oligomers of (CUG) n RNA binders by modular assembly (23,24), combinatorial chemistry (25,26), and high-throughput screening (27,28). …”

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confidence: 99%

Identification of Plant-derived Alkaloids with Therapeutic Potential for Myotonic Dystrophy Type I

Herrendorff

Faleschini

Stiefvater

et al. 2016

Journal of Biological Chemistry

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Myotonic dystrophy type I (DM1) is a disabling neuromuscular disease with no causal treatment available. This disease is caused by expanded CTG trinucleotide repeats in the 3 UTR of the dystrophia myotonica protein kinase gene. On the RNA level, expanded (CUG) n repeats form hairpin structures that sequester splicing factors such as muscleblind-like 1 (MBNL1). Lack of available MBNL1 leads to misregulated alternative splicing of many target pre-mRNAs, leading to the multisystemic symptoms in DM1. Many studies aiming to identify small molecules that target the (CUG) n -MBNL1 complex focused on synthetic molecules. In an effort to identify new small molecules that liberate sequestered MBNL1 from (CUG) n RNA, we focused specifically on small molecules of natural origin. Natural products remain an important source for drugs and play a significant role in providing novel leads and pharmacophores for medicinal chemistry. In a new DM1 mechanism-based biochemical assay, we screened a collection of isolated natural compounds and a library of over 2100 extracts from plants and fungal strains. HPLC-based activity profiling in combination with spectroscopic methods were used to identify the active principles in the extracts. The bioactivity of the identified compounds was investigated in a human cell model and in a mouse model of DM1. We identified several alkaloids, including the ␤-carboline harmine and the isoquinoline berberine, that ameliorated certain aspects of the DM1 pathology in these models. Alkaloids as a compound class may have potential for drug discovery in other RNA-mediated diseases. Myotonic dystrophy type I (DM1)4 is the most common muscular dystrophy in the adult population, with a relatively high prevalence of about 1:8000 (1). This autosomal dominantly inherited disease affects multiple organs, most prominently the skeletal muscle, with wasting, weakness, and an inability to relax (myotonia) (1). Currently, there is no effective treatment for this disabling disease. The pathomechanism of DM1 is linked to a CTG n expansion in the 3Ј UTR of the dystrophia myotonica protein kinase (DMPK) gene (2, 3), leading to a toxic gain-of-function RNA (4, 5). The mutant DMPK transcript is entrapped within nuclei of affected cells, where it forms aggregates (foci) with splicing factors such as muscleblind-like 1 (MBNL1) (6, 7). Bound to mutant DMPK (CUG) n RNA, MBNL1 is no longer available for correct splicing of its target pre-mRNAs (8, 9). Thus, the splicing of a multitude of pre-mRNAs is misregulated, including the skeletal muscle chloride channel (CLCN1), the insulin receptor (INSR), sarcoplasmic/endoplasmic reticulum Ca 2ϩ ATPase 1 (SERCA1), and cardiac troponin T type 2 (TNNT2) pre-mRNA (10 -16). Interestingly, the missplicing of some pre-mRNAs can be linked directly to a certain disease symptom, e.g. myotonia in the case of the CLCN1 pre-mRNA. MBNL1 sequestration by (CUG) n RNA causes inclusion of alternative exon 7a, leading to a shift in the open reading frame and to premature termination of translation (12, 1...

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“…Several key advances have pushed the RNA-targeting field forward including those in RNA structural biology, structure-based approaches including modeling of dynamic ensembles, and identification of RNA-binding modules (Batey et al, 2004; Childs-Disney et al, 2014; Davidson et al, 2009; Disney, 2013; Disney et al, 2014; Gallego and Varani, 2001; Jahromi et al, 2013a; Lee et al, 2010; Montange and Batey, 2006; Ofori et al, 2012; Palde et al, 2010; Parkesh et al, 2011; Shortridge and Varani, 2015; Stelzer et al, 2011; Trausch et al, 2011; Yildirim et al, 2013). High-resolution structures of ribosomes and other RNA-protein complexes combined with modeling of RNA dynamics have enabled structure-based approaches to develop new antibiotics and antivirals.…”

Section: Leveraging Rna Structure To Design Chemical Probes Of Functionmentioning

confidence: 99%

“…A number of attempts were made to optimize MTX by means of classic medicinal chemistry (Yang et al, 2009; Zheng et al, 2009) or conjugating it to aminoglycosides (Artigas and Marchán, 2015; Artigas et al, 2015). In addition, alternative chemotypes were actively sought via dynamic combinatorial chemistry (Lõpez-Senín et al, 2011; Ofori et al, 2012), and ‘Janus’-type compounds were designed to recognize the GU wobble base pair created by the mutations (Artigas and Marchán, 2013). …”

Section: Leveraging Rna Structure To Design Chemical Probes Of Functionmentioning

confidence: 99%

RNA Structures as Mediators of Neurological Diseases and as Drug Targets

Bernat

Disney

2015

Neuron

114

107

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RNAs adopt diverse folded structures that are essential for function and thus play critical roles in cellular biology. A striking example of this is the ribosome, a complex, three-dimensionally folded macromolecular machine that orchestrates protein synthesis. Advances in RNA biochemistry, structural and molecular biology, and bioinformatics have revealed other non-coding RNAs whose functions are dictated by their structure. It is not surprising that aberrantly folded RNA structures contribute to disease. In this review, we provide a brief introduction into RNA structural biology and then describe how RNA structures function in cells and cause or contribute to neurological disease. Finally, we highlight successful applications of rational design principles to provide chemical probes and lead compounds targeting structured RNAs. Based on several examples of well-characterized RNA-driven neurological disorders, we demonstrate how designed small molecules can facilitate study of RNA dysfunction, elucidating previously unknown roles for RNA in disease, and provide lead therapeutics.

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From dynamic combinatorial ‘hit’ to lead: in vitro and in vivo activity of compounds targeting the pathogenic RNAs that cause myotonic dystrophy

Cited by 74 publications

References 50 publications

Lomofungin and dilomofungin: inhibitors of MBNL1-CUG RNA binding with distinct cellular effects

Lomofungin and dilomofungin: inhibitors of MBNL1-CUG RNA binding with distinct cellular effects

Identification of Plant-derived Alkaloids with Therapeutic Potential for Myotonic Dystrophy Type I

RNA Structures as Mediators of Neurological Diseases and as Drug Targets

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